Non-fusion soluble expression of broad-spectrum antivirus protein in Escherichia coli by translational-coupling with SUMO / 军事医学

Objective To design and construct a new non-fusion soluble expression vector pTIG-mSUMO(small ubiq-uitin-related modifier) using the widely used solubility promoting protein SUMO and based on the translational coupling phenomenon in order to enable the non-fusion soluble expression of the broad-spectrum antiviral protein RA in Escherichia coli by pTIG-mSUMO.Methods The smt3 gene coding for SUMO protein was cloned from yeast genome DNA by PCR. After directed-site silent mutation to eliminate the EcoRⅠsite, the mutant mSUMO was inserted into pET-22b to obtain the translational coupling expression vector pTIG-mSUMO.The RA was subject to PCR amplification and cloned into the pTIG-mSUMO to obtain the expression plasmid pTIG-mSUMO/RA which was supposed to direct the soluble expression of RA by the translational coupling with mSUMO.Results A translational coupling expression vector pTIG-mSUMO which could di-rect/drive the SUMO and heterogonous protein non-fusion expression simultaneously was designed and constructed.The Western blotting result indicated that pTIG-mSUMO could direct the high-level expression of RA, around 40%of which was soluble.Conclusion A translational coupling expression vector pTIG-mSUMO is obtained.After coupling with SUMO, RA is highly expressed in E.coli and both the expression level and solubility are greatly improved.pTIG-mSUMO might contrib-ute to soluble expression of other proteins.

Cross-linking and immunoprecipitation assay and its development / 军事医学

It′s reported that RNA-binding proteins ( RBP) play key roles in post-transcriptional regulation of eukaryotic genes.The aberrations of RBP are associated with a large number of human disorders , particularly autoimmune and neuro-logic diseases .The interaction between RNA and proteins has been widely explored since the development of the method known as RNA immunoprecipitation with differential display or microarray analysis (RIP-ChIP) around the year of 2000. Since then, diverse derivatives of the RIP-ChIP, such as ultraviolet crosslinking and immunoprecipitation ( CLIP), high-throughput sequencing of CLIP cDNA library (HITS-CLIP), photoactivatable -ribonucleoside-enhanced crosslinking and immunoprecipitation ( PAR-CLIP) , and individual nucleotide resolution CLIP ( iCLIP ) have been developed .All these methods have some advantages over the original RIP-ChIP and greatly facilitate the study of RBP-RNA interactions .Addi-tionally , aided by the next-generation sequencing , transcriptome-wide identification of RBP target sites has become possible and the RNA-binding site resolution of RBP has also improved to some degree .We introduced the basic principles and processes of the interactions between proteins and RNA , focusing on the advantages , disadvantages and prospect of the present genome-wide version of CLIP .